Aspergillus niger and Aspergillus carbonarius in Corinth Raisin and Wine-Producing Vineyards in Greece: Population Composition, Ochratoxin A Production and Chemical Control

Vineyard surveys of Corinth raisin cultivar carried out in the Peloponnese region of Greece during 2002 and of wine‐producing grape cultivars (Cabernet Sauvignon and Grenache Rouge) on the island of Rhodes, Greece, during 2003, demonstrated the occurrence of various Aspergillus spp. in berries of bunches at harvest. Aspergillus niger and A. carbonarius were predominantly isolated from sampled berries. Although the prevailing Aspergillus spp. isolates belonged mainly to A. niger aggregate, isolates of A. carbonarius were by far the most efficient Ochratoxin A (OTA) producers as revealed by the enzyme‐linked immunosorbent assay test. This study provides the first evidence concerning the composition of Aspergillus populations in raisin and wine‐producing vineyards and offers convincing data for their ability to produce various levels of OTA in Corinth raisins and wine‐producing grapes in Greece. Furthermore, it demonstrates that chemical applications with the fungicide Switch, especially under low to intermediate Aspergillus infection of vineyards, could both significantly reduce the occurrence of OTA‐producing Aspergillus spp. and restrict sour rot severity. In contrast, vineyard applications with the fungicides Carbendazim or Chorus were ineffective in controlling the fungus in Corinth raisin cultivar.     

<Emphasis Type="Italic">Aspergillus vadensis</Emphasis>, a new species of the group of black Aspergilli

A strain from the group of black Aspergilli was analysed in detail to determine the species to which it belongs. A detailed analysis of morphology, RFLP patterns and metabolite profiles was carried out. In addition, a phylogenetic tree was constructed for the black Aspergilli using the ITS and the -tubulin sequences of the individual strains. The new species differs by its poor growth on glycerol and galacturonate and its unique extrolite profile consisting of aurasperone B, nigragillin, asperazine and kotanins. RFLP analysis using three genes as probes also resulted in a unique pattern. These data indicate that the strain was closely related but not identical to Aspergillus foetidus, Aspergillus niger and Aspergillus tubingensis. It was therefore designated as a novel species and named Aspergillus vadensis.     

Isolation and characterization of six polymorphic microsatellite loci in Aspergillus niger

Certain strains of Aspergillus niger produce ochratoxin A in food and in animal feeds. Six polymorphic microsatellite markers suitable for population analysis were developed for A. niger through screening published sequences for microsatellite repeats. Polymorphism was evaluated for 28 isolates of A. niger, including toxigenic strains. Loci displayed six to 13 alleles. Investigation of cross‐species amplifications with Aspergillus carbonarius and Aspergillus japonicus showed limited success.     

Isolation and identification of ochratoxin A-producing Aspergillus section Nigri strains from California raisins

Aims: To determine incidence and levels of ochratoxin A (OTA) in California raisins and to isolate and characterize OTA‐producing fungi from California raisin vineyard populations. Methods and Results: Forty raisin clusters sampled from four California vineyards in the San Joaquin Valley were analysed for OTA content using immunoaffinity and HPLC methods. OTA was detected in 93% of the samples, at levels from 0·06 to 11·4 ng g^?1. From these raisin samples, a total of 400 strains of Aspergillus were isolated and analysed for OTA production. Twelve isolates (3%), from five raisin samples, produced OTA. These isolates were identified as Aspergillus carbonarius, based on morphological characteristics and multilocus sequence analysis. Levels of OTA produced by these isolates on raisin agar ranged from 0·9 to 15 μg g^?1. Conclusions: OTA is a common contaminant of raisin vineyards, but average levels are much lower than EU regulatory limits for dried fruit. The primary species responsible for OTA contamination in California raisins is A. carbonarius. Significance and Impact of the Study: This study illustrates that low‐level OTA contamination of raisins occurs in California and that ecological studies of A. carbonarius within the Aspergillus section Nigri population on raisins are warranted to monitor ochratoxigenic potential of the crop.     

Effect of gentian violet on the growth of the N and T RFLP types of the Aspergillus niger aggregate

Taxa included in the Aspergillus niger aggregate are difficult to distinguish by phenotypic characterization. In this work, the effect of gentian violet on the growth of the N and T RFLP types of A. niger aggregate strains has been investigated. In total, 105 strains from different sources and origins, including reference cultures and field isolates were studied. Type N and T RFLP patterns, ochratoxin A production and the effect of different concentrations of gentian violet on the growth were determined in these strains. Forty nine strains belonged to the N type and 56 strains to the T type. Sixteen out of the 105 strains assayed were OTA producers. All the OTA-producing species belonged to the RFLP type N and none of the T type strains was able to produce OTA. Approximately 90% of the N type strains grew in the presence of 25 ppm of gentian violet. Only five N type strains did not grow on this medium. One of these strains was A. niger ATCC 22343, a well documented induced mutant strain and the remaining four strains belonged to the new species A. brasiliensis. On the contrary, all the T type strains failed to grow on this medium after 3 days of incubation (sensitivity 89.79%; specificity 100%). The use of growth in gentian violet as an additional character for classification and identification purposes in this taxonomic group may be useful because no phenotypic methods have yet been found that can distinguish between these species.     

Differential Organ Distribution,Pathogenicity and Benomyl Sensitivity of Colletotrichum spp. from Blackberry Plants in Northern Colombia

Blackberry anthracnose, caused by Colletotrichum spp., is an important disease of cultivated blackberry in the world. In Colombia, it is the number one limiting factor for commercial production. This study was conducted to determine the species of Colletotrichum infecting blackberry plants as well as the organ distribution, pathogenicity and response to benomyl of the isolated strains. Sixty isolates from stems (n = 20), thorns (n = 20) and inflorescences (n = 20) were identified as Colletotrichum acutatum and Colletotrichum gloeosporioides by a species‐specific polymerase chain reaction (PCR). Both Colletotrichum species were found in the same plant but on different organs. Colletotrichum gloeosporioides species predominated in thorn lesions (n = 16) and C. acutatum in stems (n = 15) and inflorescence (n = 15). Pathogenicity assays on detached blackberry organs demonstrated differences between the two species with an average period of lesion development of 8.7 days for C. gloeosporioides and 10.3 days for C. acutatum. Wound inoculated organs had 90% disease development compared to 17.5% in non‐wounded. All C. acutatum isolates (n = 34) were benomyl tolerant, whereas C. gloeosporioides isolates (n = 26) were 30.7% sensitive and 69.2% moderately tolerant. Phylogenetic analysis with ITS sequences of a subset of 18 strains showed that strains classified as C. gloeosporioides had 100% identity to Colletotrichum kahawae, which belongs to the C. gloeosporioides species complex, whereas C. acutatum strains clustered into two different groups, with high similarity to the A2 and the A4 molecular groups. These data demonstrate for the first time the differential distribution of both species complexes in blackberry plant organs and further clarifies the taxonomy of the strains.     

Temperature and incubation time effects on growth and ochratoxin A production by Aspergillus sclerotioniger and Aspergillus lacticoffeatus on culture media

Aims: As there is no knowledge of the influence of abiotic factors on the two new ochratoxin A (OTA)‐producing species Aspergillus sclerotioniger and Aspergillus lacticoffeatus, the aim of this study was to evaluate the effect of temperature and incubation time on growth and OTA production by these species on culture media. Methods and Results: The study was carried out on yeast extract sucrose agar (YES) and Czapek yeast extract agar (CYA) incubated at ten different temperatures from 5 to 50°C (at 5°C intervals). Growth assessment and OTA production were determined after 5, 10, 15, 20 and 30 days of incubation at each temperature. Aspergillus sclerotioniger grew from 10 to 35°C; OTA was detected from 10 to 35°C and the highest concentration was achieved at 15°C in CYA. Aspergillus lacticoffeatus grew from 10 to 45°C; OTA was detected from 15 to 45°C, and the maximum concentration was produced after 5 days at 25°C in YES. Conclusions: The studied species can produce OTA over a wide range of temperatures and significant amounts can be produced in only 5 days. Significance and Impact of the Study: This is the first report on the influence of ecophysiological factors on these two ochratoxigenic species. The pattern of effects of temperature on growth and OTA production by A. sclerotioniger and A. lacticoffeatus was similar to those reported for the closely related species Aspergillus carbonarius and Aspergillus niger, respectively. The two new OTA‐producing species have both been isolated from coffee beans, and the closely related ochratoxigenic species of section Nigri, A. carbonarius and A. niger are important sources of OTA in this substrate.     

Recombination of mitochondrial DNAs following transmission of mitochondria among incompatible strains of black Aspergilli

Successful intra- and interspecific mitochondrial transfers were performed by polyethylene glycol (PEG)-induced protoplast fusion among incompatible strains belonging to the Aspergillus niger species aggregate. The mitochondrial DNAs (mtDNAs) of the strains examined were of three main types based on their restriction fragment length polymorphism (RFLP) profiles. mtDNA types 1 and 2 correspond to A. niger and A. tubingensis species, respectively, while type 3 is represented by some Brazilian wild-type isolates (possibly a distinct species or subspecies). mtDNA types 1 and 2 could be further divided into several subgroups (1a–1e and 2a–2f ). All these strains, representing different RFLP groups or subgroups, were fully incompatible with respect to nuclear complementation. The transfer experiments were carried out under selection pressure, using a mitochondrial oligomycin-resistant mutant of mtDNA type 1a as donor. Following fusion mitochondrial oligomycin-resistant progenies were recovered in the presence of oligomycin by selecting for the nuclear phenotypes of the oligomycin-sensitive recipient strains. All attempted transfers were successful, and resulted in different varieties of resistant recombinant mitochondrial progenies at various frequencies. Within the group of strains of mtDNA type 1, the transfer of oligomycin-resistant mitochondria resulted in the appearance of a single recombinant type of RFLP profile in each case. The recombination events were more complex when the transfer of oligomycin resistance occurred between strains representing different species (mtDNA groups 1a→2 and 1a→3). A great variety of recombinant mtDNA RFLP profiles appeared. Explanation for this phenomenon are discussed on the basis of preliminary physical mapping data. Received: 16 July 1996 / Accepted: 2 December 1996     

Brief in vitro study on Botrytis cinerea and Aspergillus carbonarius regarding growth and ochratoxin A

Aims: To evaluate the effect of Botrytis cinerea growth on ochratoxin A (OTA) production by Aspergillus carbonarius and degradation. Methods and Results: OTA‐producing A. carbonarius and B. cinerea were grown on grape‐like medium at 20°C for 7 days. Radii of colonies were daily recorded and OTA was analysed. In addition, each B. cinerea isolate was inoculated on grape‐like synthetic nutrient medium (SNM) paired with each A. carbonarius isolate at a distance of 45 mm. Botrytis cinerea isolates were also grown in OTA‐spiked SNM. Growth rates of B. cinerea and A. carbonarius were 20 and 7·5 mm day^?1, respectively. The growth of the colonies of each species stopped when they contacted each other in paired cultures. OTA production by A. carbonarius in the contact area was affected by B. cinerea, but no clear trend was observed. All B. cinerea isolates showed to degrade between 24·2% and 26·7% of OTA from spiked SNM. Conclusions: The ecological advantage of B. cinerea, in terms of growth rate, vs. OTA‐producing Aspergillus in some wine‐growing regions and its ability to degrade OTA may explain the low levels of this toxin in noble wines. Significance and Impact of the Study: At determinate conditions, the presence of B. cinerea in grapes with A. carbonarius may help in reducing OTA accumulation.     

Study of Spanish Grape Mycobiota and Ochratoxin A Production by Isolates of Aspergillus tubingensis and Other Members of Aspergillus Section Nigri

The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were Alternaria, Cladosporium, and Aspergillus. The isolation frequency of Aspergillus spp. section Nigri in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of Aspergillus carbonarius and 14.3% of Aspergillus tubingensis isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No Aspergillus niger isolate had the ability to produce this toxin under the conditions assayed. Identification of the A. niger aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of A. niger CECT 2807 and A. tubingensis CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of A. tubingensis matched 99 to 100% with the nucleotide sequence of strain A. tubingensis CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of A. tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.     

Genetic variability and taxonomic revision of the genus Auxenochlorella (Shihira et Krauss) Kalina et Puncocharova (Trebouxiophyceae,Chlorophyta)

The monotypic genus Auxenochlorella with its type species A. protothecoides is so far only known from specific habitats such as the sap of several tree species. Several varieties were described according to physiological performances in culture on different organic substrates. However, two strains designated as Auxenochlorella were isolated from other habitats (an endosymbiont of Hydra viridis and an aquatic strain from an acidic volcano stream). We studied those isolates and compared them with six strains of Auxenochlorella belonging to different varieties. The integrative approach used in this study revealed that all strains showed similar morphology but differed in their SSU and ITS rDNA sequences. The Hydra endosymbiont formed a sister taxon to A. protothecoides, which included the varieties protothecoides, galactophila, and communis. The variety acidicola is not closely related to Auxenochlorella and represented its own lineage within the Trebouxiophyceae. In view of these results, we propose a new species of Auxenochlorella, A. symbiontica, for the Hydra symbiont, and a new genus Pumiliosphaera, with its type species, P. acidophila, for acidophilic strain. These results are supported by several compensatory base changes in the conserved region of ITS‐2 and ITS‐2 DNA barcodes.     

Phylogeny,morphology and pathogenicity of Elsinoë ampelina,the causal agent of grapevine anthracnose in Brazil and Australia

Anthracnose caused by Elsinoë ampelina is one of the most important table grape diseases in humid regions in Brazil and Australia. The objective of this study was to characterize E. ampelina isolates from Brazil and Australia by means of phylogenetic analyses, morphological features and pathogenicity tests. Phylogenetic relationships among 35 isolates were determined based on a data set of internal transcribed spacer (ITS), histone H3 (HIS3) and elongation factor 1‐α (TEF) sequences. In phylogenetic tree analyses, using a combined ITS and TEF sequence alignment, all E. ampelina isolates were clustered together in a single well‐supported clade. In contrast to the absence of genetic variability within ITS and TEF sequences, HIS3 sequences showed 54 polymorphic sites. The haplotype network generated from HIS3 data set showed four distinct haplotypes. EA1 was the predominant haplotype including 29 isolates from both countries. High genetic variability was observed in two Brazilian isolates, haplotype EA4, which may have lost the intron region during species evolution. Colony colours differed between Brazilian and Australian isolates, but showed similar wrinkled colony texture, absence of spores, sparse‐to‐absent white aerial mycelium and slow growth (0.049–0.060 mm/day). Brazilian isolates produced conidia of 5.65 × 2.65 μm, larger than conidia from Australian isolates, which measured 5.14 × 2.30 μm. In pathogenicity tests, all nine Australian isolates inoculated were pathogenic on detached canes and potted vines of table grape.     

Identification of Colletotrichum Species Causing Bitter Rot of Apple and Pear in Croatia

Symptoms of bitter rot were observed on apple and pear fruits in the field and in storage in Croatia between 2009 and 2011. Fifteen Colletotrichum isolates from apple and two from pear were collected and identified by sequencing of the ITS1 and ITS2 regions of the ribosomal DNA. Phylogenetic analysis revealed that ten isolates from apple and two isolates from pear could be identified as Colletotrichum fioriniae, five isolates from apple were clustered in Colletotrichum clavatum, while one isolate was in the Colletotrichum acutatum A7 group. All isolates caused typical bitter rot symptoms when inoculated on apple and pear fruits.     

Identification of ochratoxin A producing Aspergillus carbonarius and A. niger clade isolated from grapes using the loop‐mediated isothermal amplification (LAMP) reaction

Aims

To develop two assays based on the loop‐mediated isothermal amplification (LAMP) of DNA for the quick and specific identification of Aspergillus carbonarius and ochratoxigenic strains of the Aspergillus niger clade isolated from grapes.

Methods and Results

Two sets of primers were designed based on the polyketide synthase genes involved or putatively involved in ochratoxin A (OTA) biosynthesis in A. carbonarius and A. niger clade. Hydroxynaphthol blue was used as indirect method to indicate DNA amplification. The limit of detection of both assays was comparable to that of a PCR reaction. Specificities of the reactions were tested using DNA from different black aspergilli isolated from grapes. The two LAMP assays were then used to identify A. carbonarius and ochratoxigenic A. niger and A. awamori grown in pure cultures without a prior DNA extraction.

Conclusions

The two LAMP assays permitted to quickly and specifically identify DNA from OTA‐producing black aspergilli, as well as isolates grown in pure culture.

Significance and Impact of the Study

Monitoring vineyards for the presence of OTA‐producing strains is part of the measures to minimize the occurrence of OTA in grape products. The two LAMP assays developed here could be potentially used to speed the screening process of vineyards for the presence of OTA‐producing black aspergilli.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Molecular diagnosis of ochratoxinogenicAspergillus species

Toxigenic and non-toxigenic black aspergilli belonging to theAspergillus niger aggregate and toA. carbonarius were compared to each other and to strains of other species by DNA fingerprinting. AFLPs showed a clear separation ofA. niger andA. carbonarius. However, no clear correlation between the genetic similarity of the strains and the ability to produce ochratoxin A (OTA) was detected. Based on AFLP, marker sequences were chosen for the construction of SCAR-PCR primers for the detection ofA. carbonarius. A similar approach was used forA. ochraceus, another fungus of concern regarding ochratoxin A contamination of coffee. Cluster analysis ofA. ochraceus isolates mainly from Brazilian coffee showed a very close genetic similarity. Three species specific primer pairs were developed and one of these was used for the PCR and realtime PCR (RT-PCR) based detection of the mould in green coffee.A. ochraceus was specifically and rapidly detected and quantified in green coffee. A positive correlation between the amount of DNA and OTA content was established.     

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH‐psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity‐based methods and tree‐based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH‐psbA. The ITS provided better results with 30.61–38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree‐based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.     

Occurrence of Ochratoxin A-Producing Fungi in Commercial Corn Kernels in Argentina

Potentially ochratoxigenic Aspergillus and Penicillium species were identified and the natural occurrence of ochratoxin A (OTA) in corn kernels was evaluated. Likewise, the capacity to produce OTA by Aspergillus section Nigri and Circumdati was investigated. A total of 50 corn samples for human consumption was collected in the south of Córdoba Province. The surface-disinfected method for mycobiota determination was used. The OTA detection was performed by HPLC. OTA production was tested in strains belonging to section Nigri and Circumdati. Statistical analysis demonstrated that the specie A. flavus was isolated in higher frequency (p<0.01) from corn kernels in DRBC and DG18 media. The percentage of corn kernels contaminated by A. niger var. niger was similar in DRBC and DG18 media. The frequency of grains contaminated by A. flavus and A. niger var. awamori was higher than A. niger var. niger and A. japonicus var. japonicus (p<0.01) in DG18 media. The other potentially ochratoxigenic species, A. ochraceus, was isolated between 5% and 10% of the corn kernels in DG18 and DRBC media, respectively. The OTA producing species P. verrucosum was not isolated. All samples of corn were OTA negative (<1 ng g⁻¹). Thirty strains (25%) of the black Aspergillus were OTA producers. From four strains of A. ochraceus isolated, only one produced OTA. Due to the storage variable conditions could not be adequate in this substrate, the presence of ochratoxigenic strains of section Nigri and OTA needs to be evaluated for a longer time to establish the toxicological risk for human beings. The contamination of stored corn kernels with A. flavus and Aspergillus section Nigri was significant.     

Impact of some environmental factors on growth and production of ochratoxin A of/by <Emphasis Type="Italic">Aspergillus tubingensis,A. niger</Emphasis>, and <Emphasis Type="Italic">A. carbonarius</Emphasis> isolated from moroccan grapes

The effects of temperature, water activity (a_w), incubation time, and their combinations on radial growth and ochratoxin A (OTA) production of/by eight Aspergillus niger aggregate strains (six A. tubingensis and two A. niger) and four A. carbonarius isolated from Moroccan grapes were studied. Optimal conditions for the growth of most studied strains were shown to be at 25°C and 0.95 a_w. No growth was observed at 10°C regardless of the water activity and isolates. The optimal temperature for OTA production was in the range of 25°C∼30°C for A. carbonarius and 30°C∼37°C for A. niger aggregate. The optimal a_w for toxin production was 0.95∼0.99 for A. carbonarius and 0.90∼0.95 for A. niger aggregate. Mean OTA concentration produced by all the isolates of A. niger aggregate tested at all sampling times shows that maximum amount of OTA (0.24 μg/g) was produced at 37°C and 0.90 a_w. However, for A. carbonarius, mean maximum amounts of OTA (0.22 μg/g) were observed at 25°C and 0.99 a_w. Analysis of variance showed that the effects of all single factors (a_w, isolate, temperature and incubation time) and their interactions on growth and OTA production were highly significant.